This application is a 371 of PCT/GB00/03303 filed Aug. 30, 2000.
The present invention relates to compounds which elevate pyruvate dehydrogenase (PDH) activity, processes for their preparation, pharmaceutical compositions containing them as active ingredient, methods for the treatment of disease states associated with reduced PDH activity, to their use as medicaments and to their use in the manufacture of medicaments for use in the elevation of PDH activity in warm-blooded animals such as humans, in particular the treatment of diabetes mellitus, peripheral vascular disease and myocardial ischaemia in warm-blooded animals such as humans, more particularly to their use in the manufacture of medicaments for use in the treatment of diabetes mellitus in warm-blooded animals such as humans.
Within tissues adenosine triphosphate (ATP) provides the energy for synthesis of complex molecules and, in muscle, for contraction. ATP is generated from the breakdown of energy-rich substrates such as glucose or long chain free fatty acids. In oxidative tissues such as muscle the majority of the ATP is generated from acetyl CoA which enters the citric acid cycle, thus the supply of acetyl CoA is a critical determinant of ATP production in oxidative tissues. Acetyl CoA is produced either by xcex2-oxidation of fatty acids or as a result of glucose metabolism by the glycolytic pathway. The key regulatory enzyme in controlling the rate of acetyl CoA formation from glucose is PDH which catalyses the oxidation of pyruvate to acetyl CoA and carbon dioxide with concomitant reduction of nicotinamide adenine dinucleotide (NAD) to NADH.
In disease states such as both non-insulin dependent (NIDDM) and insulin-dependent diabetes mellitus (IDDM), oxidation of lipids is increased with a concomitant reduction in utilisation of glucose, which contributes to the hyperglycaemia. Reduced glucose utilisation in both IDDM and NIDDM is associated with a reduction in PDH activity. In addition, a further consequence of reduced PDH activity may be that an increase in pyruvate concentration results in increased availability of lactate as a substrate for hepatic gluconeogenesis. It is reasonable to expect that increasing the activity of PDH could increase the rate of glucose oxidation and hence overall glucose utilisation, in addition to reducing hepatic glucose output. Another factor contributing to diabetes mellitus is impaired insulin secretion, which has been shown to be associated with reduced PDH activity in pancreatic xcex2-cells (in a rodent genetic model of diabetes mellitus Zhou et al. (1996) Diabetes 45:580-586).
Oxidation of glucose is capable of yielding more molecules of ATP per mole of oxygen than is oxidation of fatty acids. In conditions where energy demand may exceed energy supply, such as myocardial ischaemia, intermittent claudication, cerebral ischaemia and reperfusion, (Zaidan et al., 1998; J. Neurochem. 70:233-241), shifting the balance of substrate utilisation in favour of glucose metabolism by elevating PDH activity may be expected to improve the ability to maintain ATP levels and hence function.
An agent which is capable of elevating PDH activity may also be expected to be of benefit in treating conditions where an excess of circulating lactic acid is manifest such as in certain cases of sepsis.
The agent dichloroacetic acid (DCA) which increases the activity of PDH after acute administration in animals, (Vary et al., 1988; Circ. Shock, 24:3-18), has been shown to have the predicted effects in reducing glycaemia, (Stacpoole et al., 1978; N. Engl. J. Med. 298:526-530), and as a therapy for myocardial ischaemia (Bersin and Stacpoole 1997; American Heart Journal, 134:841-855) and lactic acidaemia, (Stacpoole et al., 1983; N. Engl. J. Med. 309:390-396).
PDH is an intramitochondrial multienzyme complex consisting of multiple copies of several subunits including three enzyme activities E1, E2 and E3, required for the completion of the conversion of pyruvate to acetyl CoA (Patel and Roche 1990; FASEB J., 4:3224-3233). E1 catalyses the non-reversible removal of CO2 from pyruvate; E2 forms acetyl CoA and E3 reduces NAD to NADH. Two additional enzyme activities are associated with the complex: a specific kinase which is capable of phosphorylating E1 at three serine residues and a loosely-associated specific phosphatase which reverses the phosphorylation. Phosphorylation of a single one of the three serine residues renders the E1 inactive. The proportion of the PDH in its active (dephosphorylated) state is determined by a balance between the activity of the kinase and phosphatase. The activity of the kinase may be regulated in vivo by the relative concentrations of metabolic substrates such as NAD/NADH, CoA/acetylCoA and adenine diphosphate (ADP)/ATP as well as by the availability of pyruvate itself.
European Patent Publication No. 625516 refers to compounds which are capable of relaxing bladder smooth muscle and which may be used in the treatment of urge incontinence. We have found, surprisingly, that compounds also containing a sulphonamide moiety disclosed in the present invention are very good at elevating PDH activity, a property nowhere disclosed in EP 625516. The present invention is based on the surprising discovery that certain compounds elevate PDH activity, a property of value in the treatment of disease states associated with disorders of glucose utilisation such as diabetes mellitus, obesity, (Curto et al., 1997; Int. J. Obes. 21:1137-1142), and lactic acidaemia. Additionally the compounds may be expected to have utility in diseases where supply of energy-rich substrates to tissues is limiting such as peripheral vascular disease, (including intermittent claudication), cardiac failure and certain cardiac myopathies, muscle weakness, hyperlipidaemias and atherosclerosis (Stacpoole et al., 1978; N. Engl. J. Med. 298:526-530). A compound that activates PDH may also be useful in treating Alzheimer disease (AD) (J Neural Transm (1998) 105:855-870).
Accordingly, the present invention provides a compound of formula (I): 
wherein:
R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, naphthyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C2-6alkenyl, C2-6alkynyl, C1-6alkyl optionally substituted by one or more Q, R8Txe2x80x94, R9C1-6alkylTxe2x80x94 and a heterocyclic group optionally substituted on a ring carbon by one or more Q and wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D,
or R1 and R2 together with the nitrogen atom to which they are attached form a group Het which is optionally substituted on a ring carbon by one or more Q and wherein if group Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D;
R3 and R4 are independently Ckalkyl optionally substituted by 1 to 2k+1 atoms selected from fluoro and chloro wherein k is 1-3;
or R3 and R4 together with the carbon atom to which they are attached, form a Cmcycloalkyl ring optionally substituted by 1 to 2m-2 fluorine atoms wherein m is 3-5;
R5 is hydrogen, C1-6alkyl, haloC1-6alkyl, C1-6alkoxy, haloC1-6alkoxy, cyano, nitro, C2-6alkenyloxy, trifluoromethylthio, halo, hydroxy, amino, Nxe2x80x94(C1-6alkyl)amino, Nxe2x80x94(C1-6alkyl)2amino, C1-6alkanoylamino, C1-6alkanoyl(Nxe2x80x94C1-6alkyl)amino, C1-6alkylsulphonylamino, C1-6alkylsulphonyl(Nxe2x80x94C1-6alkyl)amino, thiol, C1-6alkylsulphanyl, C1-6alkylsulphinyl, C1-6alkylsulphonyl, sulphamoyl, Nxe2x80x94(C1-6alkyl)aminosulphonyl, Nxe2x80x94(C1-6alkyl)2aminosulphonyl, carboxy, carbamoyl, Nxe2x80x94(C1-6alkyl)carbamoyl, Nxe2x80x94(C1-6alkyl)2carbamoyl, C1-6alkoxycarbonyl, formyl, C1-6alkanoyl, C1-6alkanoyloxy, C2-6alkenyl or C2-6alkynyl;
R6 is hydrogen or Q;
R7 is hydrogen, trifluoromethyl, C1-4alkyl, halo, hydroxy, trifluoromethoxy, cyano, C1-4alkoxy, formyl, C1-4alkanoyl, C1-4alkanoyloxy, amino, Nxe2x80x94(C1-4alkyl)amino, Nxe2x80x94(C1-4alkyl)2amino, C1-4alkanoylamino, C1-4alkanoyl(Nxe2x80x94C1-4alkyl)amino, nitro, carboxy, carbamoyl, ureido, C1-4alkoxycarbonyl, thiol, C1-4alkylsulphanyl, C1-4alkylsulphinyl, C1-4alkylsulphonyl, sulphamoyl, Nxe2x80x94(C1-4alkyl)aminosulphonyl, Nxe2x80x94(C1-4alkyl)2aminosulphonyl, Nxe2x80x2xe2x80x94(C1-4alkyl)ureido or Nxe2x80x2xe2x80x94(C1-4alkyl)2ureido;
R8 is C1-6alkyl optionally substituted by one or more R10, C3-6cycloalkyl optionally substituted by one or more R10, phenyl optionally substituted by one or more R10, naphthyl optionally substituted by one or more R10 or a heterocyclic group optionally substituted on a ring carbon by one or more R10 and if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D;
R9 is phenyl optionally substituted by one or more R10, naphthyl optionally substituted by one or more R10 or a heterocyclic group optionally substituted on a ring carbon by one or more R10 and if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D;
R10 is C1-6alkyl-Mxe2x80x94 optionally substituted by R16, halo, hydroxy, cyano, formyl, amino, nitro, carboxy, carbamoyl, thiol, sulphamoyl, phenyl optionally substituted by R16, a heterocyclic group optionally substituted on a ring carbon by R16 wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D;
Q is C1-6alkyl-Mxe2x80x94 optionally substituted with one or more R11, C2-6alkenyl-Mxe2x80x94 optionally substituted by one or more R11, C2-6alkynyl-Mxe2x80x94 optionally substituted by one or more R11, R13xe2x80x94Mxe2x80x94, hydroxy, halo, cyano, thiol, sulphamoyl, nitro, carboxy, amino, carbamoyl, formyl or ureido;
T is xe2x80x94Oxe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94NHxe2x80x94, xe2x80x94xe2x80x94N(Nxe2x80x94C1-6alkyl)-, xe2x80x94C(O)NHxe2x80x94, xe2x80x94NHC(O)xe2x80x94, xe2x80x94C(O)N(Nxe2x80x94C1-6alkyl)-, xe2x80x94N(Nxe2x80x94C1-6alkyl)C(O)xe2x80x94, xe2x80x94SO2xe2x80x94, xe2x80x94C(S)xe2x80x94, xe2x80x94C(S)NHxe2x80x94, xe2x80x94NHC(S)xe2x80x94, xe2x80x94C(S)N(Nxe2x80x94C1-6alkyl)- or xe2x80x94N(Nxe2x80x94C1-6alkyl)C(S)xe2x80x94;
M is xe2x80x94Oxe2x80x94, xe2x80x94N(R14)xe2x80x94, xe2x80x94C(O)xe2x80x94, xe2x80x94N(R14)C(O)xe2x80x94, xe2x80x94C(O)N(R14)xe2x80x94, xe2x80x94S(O)nxe2x80x94 wherein n is 0-2, xe2x80x94OC(O)xe2x80x94, xe2x80x94C(O)Oxe2x80x94, xe2x80x94N(R14)C(O)Oxe2x80x94, xe2x80x94OC(O)N(R14)xe2x80x94, xe2x80x94C(S)N(R14)xe2x80x94, xe2x80x94N(R14)C(S)xe2x80x94, xe2x80x94SO2N(R14)xe2x80x94, xe2x80x94N(R14)SO2xe2x80x94, xe2x80x94N(R14)C(O)N(R14)xe2x80x94, xe2x80x94N(R14)C(S)N(R14)xe2x80x94, xe2x80x94SO2NHC(O)xe2x80x94, xe2x80x94SO2N(R14)C(O)xe2x80x94, xe2x80x94C(O)NHSO2xe2x80x94, xe2x80x94C(O)N(R14)SO2xe2x80x94 or M is a direct bond;
D is C1-6alkyl, C1-6alkanoyl, C1-6alkylsulphonyl, C1-6alkoxycarbonyl, carbamoyl, Nxe2x80x94(C1-6alkyl)carbamoyl, Nxe2x80x94(C1-6alkyl)2carbamoyl, benzoyl, (heterocyclic group)carbonyl, phenylsulphonyl, (heterocyclic group)sulphonyl, phenyl or a carbon linked heterocyclic group, and wherein any C1-6alkyl group may be optionally substituted by one or more R11, and wherein any phenyl or heterocyclic group may be optionally substituted on a ring carbon by one or more groups selected from R10 and if a heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from E;
E is C1-6alkyl, C1-6alkanoyl, C1-6alkylsulphonyl, C1-6alkoxycarbonyl, carbamoyl, Nxe2x80x94(C1-6alkyl)carbamoyl, Nxe2x80x94(C1-6alkyl)2carbamoyl, C1-6alkoxyC1-6alkanoyl, phenylC1-6alkyl, benzoyl, phenylC1-6alkanoyl, phenylC1-6alkoxycarbonyl or phenylsulphonyl.
R11 is hydroxy, nitro, cyano, thiol, halo, ureido, amino, Nxe2x80x94(C1-6alkyl)amino, Nxe2x80x94(C1-6alkyl)2amino, carboxy, C1-6alkoxy, carbamoyl, Nxe2x80x94(C1-6alkyl)carbamoyl, Nxe2x80x94(C1-6alkyl)2carbamoyl, formyl, sulphamoyl, Nxe2x80x94C1-6alkylaminosulphonyl, Nxe2x80x94(C1-6alkyl)2aminosulphonyl, C1-6alkylsulphonylamino, C1-6alkanoylamino, C1-6alkylsulphanyl, C1-6alkylsulphinyl, C1-6alkylsulphonyl, phenyl optionally substituted by one or more R10, naphthyl optionally substituted by one or more R10, or a heterocyclic group optionally substituted on a ring carbon by one or more R10 and if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D;
R13 is phenyl optionally substituted by one or more R10, C3-6cycloalkyl optionally substituted by one or more R10, naphthyl optionally substituted by one or more R10, or a heterocyclic group optionally substituted on a ring carbon by one or more R10 and if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D;
R14 is hydrogen or C1-6alkyl optionally substituted by one or more R15 with the proviso that R15 is not a substituent on the carbon attached to the nitrogen atom of M;
R15 is halo, hydroxy, amino, cyano, nitro, trifluoromethyl, trifluoromethoxy, C1-6alkyl, C2-6alkenyl, C2-6alkynyl, Nxe2x80x94(C1-6alkyl)amino, Nxe2x80x94(C1-6alkyl)2amino, C1-6alkanoylamino, C1-6alkanoyl(Nxe2x80x94C1-6alkyl)amino, C1-6alkylsulphonylamino, C1-6alkylsulphonyl(Nxe2x80x94C1-6alkyl)amino, thiol, C1-6alkylsulphanyl, C1-6alkylsulphinyl, C1-6alkylsulphonyl, sulphamoyl, Nxe2x80x94(C1-6alkyl)aminosulphonyl, Nxe2x80x94(C1-6alkyl)2aminosulphonyl, carboxy, carbamoyl, Nxe2x80x94(C1-6alkyl)carbamoyl, Nxe2x80x94(C1-6alkyl)2carbamoyl, C1-6alkoxycarbonyl, C1-6alkanoyl or formyl;
R16 is hydroxy, halo, cyano, C1-6alkoxy, formyl, C1-6alkanoyl, C1-6alkanoyloxy, amino, Nxe2x80x94(C1-6alkyl)amino, Nxe2x80x94(C1-6alkyl)2amino, C1-6alkanoylamino, C1-6alkanoyl(Nxe2x80x94C1-6alkyl)amino, carboxy, carbamoyl, C1-6alkoxycarbonyl, C1-6alkylsulphanyl, C1-6alkylsulphinyl, C1-6alkylsulphonyl, sulphamoyl, Nxe2x80x94(C1-6alkyl)aminosulphonyl or Nxe2x80x94(C1-6alkyl)2aminosulphonyl;
and wherein if one of R5, R6 and R7 is hydrogen the others cannot be hydrogen;
or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
In this specification the term xe2x80x9calkylxe2x80x9d includes both straight and branched chain alkyl groups but references to individual alkyl groups such as xe2x80x9cpropylxe2x80x9d are specific for the straight chain version only. For example, xe2x80x9cC1-6alkylxe2x80x9d includes C1-4alkyl, propyl, isopropyl and t-butyl. However, references to individual alkyl groups such as xe2x80x98propylxe2x80x99 are specific for the straight chained version only and references to individual branched chain alkyl groups such as xe2x80x98isopropylxe2x80x99 are specific for the branched chain version only. A similar convention applies to other radicals, for example xe2x80x9chaloC1-6alkylxe2x80x9d includes haloC1-4alkyl, trifluoromethyl, 1-chloroethyl and 2-fluoroethyl. The term xe2x80x9chaloxe2x80x9d refers to fluoro, chloro, bromo and iodo. Where a phrase such as xe2x80x9cany C1-6alkyl group may be optionally substituted by one or more groupsxe2x80x9d for the avoidance of doubt, it is to be understood that this refers to all groups that contain a C1-6alkyl group, for example this phrase would also relate to a C1-6alkanoyl group if that was listed in the paragraph.
A xe2x80x9cheterocyclic groupxe2x80x9d is a saturated, partially saturated or unsaturated, mono or bicyclic ring containing 4-12 atoms of which at least one atom is chosen from nitrogen, sulphur or oxygen, which may, unless otherwise specified, be carbon or nitrogen linked, wherein a xe2x80x94CH2xe2x80x94 group can optionally be replaced by a xe2x80x94C(O)xe2x80x94, and a ring nitrogen and/or a ring sulphur atom may be optionally oxidised to form an N- and/or an S-oxide. Suitable values for a xe2x80x9cheterocyclic groupxe2x80x9d include azetidinyl, morpholino, piperidyl, pyridyl, pyranyl, pyrrolyl, isothiazolyl, indolyl, quinolyl, thienyl, 1,3-benzodioxolyl, thiadiazolyl, piperazinyl, thiazolidinyl, pyrrolidinyl, thiomorpholino, pyrrolinyl, homopiperazinyl, tetrahydropyranyl, imidazolyl, pyrimidyl, pyrazinyl, pyridazinyl, isoxazolyl, 4-pyridone, 1-isoquinolone, 2-pyrrolidone and 4-thiazolidone.
A xe2x80x9ccarbon-linked 5-or 6-membered heteroaryl ring containing 1-3 heteroatomsxe2x80x9d is a fully unsaturated monocyclic ring containing 5 or 6 atoms of which 1-3 are chosen from nitrogen, sulphur or oxygen. Suitable values for xe2x80x9ca carbon-linked 5-or 6-membered heteroaryl ring containing 1-3 heteroatomsxe2x80x9d include pyridyl, pyrimidyl, pyrazinyl, pyridadzinyl, pyrrolyl, furyl, thienyl, pyrazolyl, imidazolyl and triazolyl.
A xe2x80x9ca carbon-linked 6-membered heteroaryl ring containing 1-2 nitrogen atomsxe2x80x9d is a fully unsaturated monocyclic ring containing 6 atoms of which 1-2 are nitrogen. Suitable values for xe2x80x9ca carbon-linked 6-membered heteroaryl ring containing 1-2 nitrogen atomsxe2x80x9d include pyridyl, pyrimidyl, pyrazinyl and pyridadzinyl.
xe2x80x9cHetxe2x80x9d is a saturated, partially saturated or fully unsaturated, mono or bicyclic ring containing 4-12 atoms, one atom of which is the nitrogen atom to which R1 and R2 are attached to, and the other atoms are either all carbon atoms or they are carbon atoms and 1-3 heteroatoms chosen from nitrogen, sulphur or oxygen, wherein a xe2x80x94CH2xe2x80x94 group can optionally be replaced by a xe2x80x94C(O)xe2x80x94, and a ring nitrogen and/or a ring sulphur atom may be optionally oxidised to form an N- and/or an S-oxide. It will be appreciated that where R1 and R2 together with the nitrogen atom to which they are attached form a group Het this nitrogen atom is not quaternised, i.e. a neutral compound is formed. Suitable values for xe2x80x9ca group Hetxe2x80x9d include azetidinyl, morpholino, piperidyl, piperazinyl, pyrrolidinyl, thiomorpholino, pyrrolinyl, homopiperazinyl, pyrrolyl, pyrazolyl, pyrazolinyl, imidazolyl, imidazolinyl, imidazolidinyl, pyrazolidinyl and triazolyl. Preferably xe2x80x9ca group Hetxe2x80x9d is morpholino, piperidyl, piperazinyl, pyrrolidinyl, thiomorpholino, pyrrolinyl or homopiperazinyl.
xe2x80x9cSaturated Hetxe2x80x9d is a saturated mono or bicyclic ring containing 5-7 atoms, one atom of which is the nitrogen atom to which R1 and R2 are attached to, and the other atoms are either all carbon atoms or they are carbon atoms and 1 nitrogen atom, wherein a xe2x80x94CH2xe2x80x94 group can optionally be replaced by a xe2x80x94C(O)xe2x80x94. It will be appreciated that where R1 and R2 together with the nitrogen atom to which they are attached form a group xe2x80x9csaturated Hetxe2x80x9d this nitrogen atom is not quaternised, i.e. a neutral compound is formed. Suitable values for xe2x80x9csaturated Hetxe2x80x9d include morpholino, piperidyl, piperazinyl, pyrrolidinyl, thiomorpholino, homopiperazinyl, imidazolidinyl and pyrazolidinyl. Preferably xe2x80x9csaturated Hetxe2x80x9d is morpholino, piperidyl or piperazinyl.
Examples of xe2x80x9cC1-6alkanoyloxyxe2x80x9d are C1-4alkanoyloxy and acetoxy. Examples of xe2x80x9cC1-6alkoxycarbonylxe2x80x9d include C1-4alkoxycarbonyl, methoxycarbonyl, ethoxycarbonyl, n- and t-butoxycarbonyl. Examples of xe2x80x9cC1-6alkoxyxe2x80x9d include C1-4alkoxy, methoxy, ethoxy and propoxy. Examples of xe2x80x9cC1-6alkanoylaminoxe2x80x9d include C1-4alkanoylamino, acetamido and propionylamino. Examples of xe2x80x9cC1-6alkylsulphanylxe2x80x9d include C1-4alkylsulphanyl, methylthio and ethylthio. Examples of xe2x80x9cC1-6alkylsulphinylxe2x80x9d include C1-4alkylsulphinyl, methylsulphinyl and ethylsulphinyl. Examples of xe2x80x9cC1-6alkylsulphonylxe2x80x9d include C1-4alkylsulphonyl, mesyl and ethylsulphonyl. Examples of xe2x80x9cC1-6alkanoylxe2x80x9d include C1-4alkanoyl, propionyl and acetyl. Examples of xe2x80x9cNxe2x80x94(C1-6alkyl)aminoxe2x80x9d include Nxe2x80x94(C1-4alkyl)amino, methylamino and ethylamino. Examples of xe2x80x9cNxe2x80x94(C1-6alkyl)2aminoxe2x80x9d include Nxe2x80x94(C1-4alkyl)2amino, di-N-methylamino, di-(N-ethyl)amino and N-ethyl-N-methylamino. Examples of xe2x80x9cC2-6alkenyloxyxe2x80x9d are C2-4alkenyloxy, vinyloxy and allyloxy. Examples of xe2x80x9cC3-6cycloalkylxe2x80x9d are cyclopropyl and cyclohexyl. Examples of xe2x80x9cC2-6alkenylxe2x80x9d are C2-4alkenyl, vinyl, allyl and 1-propenyl. Examples of xe2x80x9cC2-6alkynylxe2x80x9d are C2-4alkynyl, ethynyl, 1-propynyl and 2-propynyl. Examples of xe2x80x9chaloC1-6alkoxyxe2x80x9d are haloC1-4alkoxy, trifluoromethoxy, 2-fluoroethoxy and 1-bromopropoxy. Examples of xe2x80x9cC1-6alkanoyl(Nxe2x80x94C1-6alkyl)aminoxe2x80x9d are C1-4alkanoyl(Nxe2x80x94C1-4alkyl)amino and (N-propyl)acetamido. Examples of xe2x80x9cC1-6alkylsulphonylaminoxe2x80x9d are methylsulphonylamino and ethylsulphonylamino. Examples of xe2x80x9cC1-6alkylsulphonyl(Nxe2x80x94C1-6alkyl)aminoxe2x80x9d are C1-4alkylsulphonyl(Nxe2x80x94C1-4alkyl)amino, methylsulphonyl-(N-ethyl)-amino and ethylsulphonyl-(N-butyl)-amino. Examples of xe2x80x9cNxe2x80x94(C1-6alkyl)aminosulphonylxe2x80x9d are Nxe2x80x94(C1-4alkyl)aminosulphonyl, N-(methyl)aminosulphonyl and N-(ethyl)aminosulphonyl. Examples of xe2x80x9cNxe2x80x94(C1-6alkyl)2aminosulphonylxe2x80x9d are Nxe2x80x94(C1-4alkyl)2aminosulphonyl, N-(dimethyl)aminosulphonyl and N-(methyl)-N-(ethyl)aminosulphonyl. Examples of xe2x80x9cNxe2x80x94(C1-6alkyl)carbamoylxe2x80x9d are Nxe2x80x94(C1-4alkyl)carbamoyl, methylaminocarbonyl and ethylaminocarbonyl. Examples of xe2x80x9cNxe2x80x94(C1-6alkyl)2carbamoylxe2x80x9d are Nxe2x80x94(C1-4alkyl)2carbamoyl, dimethylaminocarbonyl and methylethylaminocarbonyl. Examples of xe2x80x9cNxe2x80x2xe2x80x94(C1-4alkyl)ureidoxe2x80x9d are Nxe2x80x2-methylureido and Nxe2x80x2-ethylureido. Examples of xe2x80x9cNxe2x80x2xe2x80x94(C1-4alkyl)2ureidoxe2x80x9d are Nxe2x80x2,Nxe2x80x2-dimethylureido and Nxe2x80x2-methyl-Nxe2x80x2-ethylureido. Examples of xe2x80x9cC1-6alkanoylaminosulphonylxe2x80x9d are C1-4alkanoylaminosulphonyl, acetylaminosulphonyl and propionylaminosulphonyl. Examples of xe2x80x9cC1-6alkanoyl(Nxe2x80x94C1-6alkyl)aminosulphonylxe2x80x9d are C1-4alkanoyl(Nxe2x80x94C1-4alkyl)aminosulphonyl, acetyl(N-methyl)aminosulphonyl and propionyl(N-ethyl)aminosulphonyl. Examples of xe2x80x9cC1-6alkylsulphonylaminocarbonylxe2x80x9d are C1-4alkylsulphonylaminocarbonyl, mesylaminocarbonyl and ethanesulphonylaminocarbonyl. Examples of xe2x80x9cC1-6alkylsulphonyl(Nxe2x80x94C1-6alkyl)aminocarbonylxe2x80x9d are C1-4alkylsulphonyl(Nxe2x80x94C1-4alkyl)aminocarbonyl, N-(methyl)mesylaminocarbonyl and N-(methyl)ethanesulphonylaminocarbonyl. Examples of xe2x80x9c(heterocyclic group)carbonylxe2x80x9d are pyridylcarbonyl and pyrimidylcarbonyl. Examples of xe2x80x9c(heterocyclic group)sulphanylxe2x80x9d are pyridylsulphanyl and pyrimidylsulphanyl. Examples of xe2x80x9c(heterocyclic group)sulphinylxe2x80x9d are pyridylsulphinyl and pyrimidylsulphinyl. Examples of xe2x80x9c(heterocyclic group)sulphonylxe2x80x9d are pyridylsulphonyl and pyrimidylsulphonyl. Examples of xe2x80x9cN-(heterocyclic group)aminoxe2x80x9d are N-pyridylamino and N-pyrimidylamino. Examples of xe2x80x9cN-(heterocyclic group)carbonylaminoxe2x80x9d are N-pyridylcarbonylamino and N-pyrimidylcarbonylamino. Examples of xe2x80x9c(heterocyclic group)sulphonylaminoxe2x80x9d are pyridylsulphonylamino and pyrimidylsulphonylamino. Examples of xe2x80x9cN-(heterocyclic group)aminosulphonylxe2x80x9d are N-pyridylaminosulphonyl and N-pyrimidylaminosulphonyl. Examples of xe2x80x9cN-(heterocyclic group)-Nxe2x80x94(C1-4alkyl)aminosulphonylxe2x80x9d are N-pyridyl-N-methylaminosulphonyl and pyrimidyl-N-ethylaminosulphonyl. Examples of xe2x80x9cN-(heterocyclic group)carbamoylxe2x80x9d are N-pyridylcarbamoyl and N-pyrimidylcarbamoyl. Examples of xe2x80x9cN-(heterocyclic group)-Nxe2x80x94(C1-4alkyl)carbamoylxe2x80x9d are N-pyridyl-N-methylcarbamoyl and N-pyrimidyl-N-ethylcarbamoyl. Examples of xe2x80x9cN-phenyl-Nxe2x80x94(C1-4alkyl)aminosulphonylxe2x80x9d are N-phenyl-N-methylaminosulphonyl and N-phenyl-N-ethylaminosulphonyl. Examples of xe2x80x9cphenylC1-4alkanoylaminoxe2x80x9d include phenylacetamido and phenylpropionylamino. Examples of xe2x80x9cN-phenyl-Nxe2x80x94(C1-4alkyl)carbamoylxe2x80x9d are N-phenyl-N-methylcarbamoyl and N-phenyl-N-ethylcarbamoyl.
Preferred values of R1, R2, R3, R4, R5, R6 and R7 are as follows. Such values may be used where appropriate with any of the definitions, claims or embodiments defined hereinbefore or hereinafter.
Preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C1-6alkyl optionally substituted by one or more Q, R8Txe2x80x94 and a heterocyclic group optionally substituted on a ring carbon by one or more Q and wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D,
or R1 and R2 together with the nitrogen atom to which they are attached form a group Het which is optionally substituted on a ring carbon by one or more Q and wherein if group Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D.
More preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C1-4alkyl optionally substituted by one or more Q, R8Txe2x80x94 and a carbon-linked 5-or 6-membered heteroaryl ring containing 1-3 heteroatoms optionally substituted by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a saturated Het which is optionally substituted on a ring carbon by one or more Q and wherein if said saturated Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D.
More preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C1-4alkyl optionally substituted by one or more Q, R8Txe2x80x94 and a carbon-linked 6-membered heteroaryl ring containing 1-2 nitrogen atoms optionally substituted by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a saturated Het which is optionally substituted on a ring carbon by one or more Q and wherein if said saturated Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D.
In another aspect of the invention, preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-6alkyl optionally substituted by one or more Q, R8Txe2x80x94 and a heterocyclic group optionally substituted on a ring carbon by one or more Q and wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D,
or R1 and R2 together with the nitrogen atom to which they are attached form a group Het which is optionally substituted on a ring carbon by one or more Q and wherein if group Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D.
In another aspect of the invention, more preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-4alkyl optionally substituted by one or more Q, R8Txe2x80x94 and a carbon-linked 5-or 6-membered heteroaryl ring containing 1-3 heteroatoms optionally substituted by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a saturated Het which is optionally substituted on a ring carbon by one or more Q and wherein if said saturated Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D.
In another aspect of the invention, particularly R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-4alkyl optionally substituted by one or more Q, R8Txe2x80x94 and a carbon-linked 6-membered heteroaryl ring containing 1-2 nitrogen atoms optionally substituted by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a saturated Het which is optionally substituted on a ring carbon by one or more Q and wherein if said saturated Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from D.
In a further aspect of the invention, preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-6alkyl optionally substituted by one or more Q and a heterocyclic group optionally substituted on a ring carbon by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a group Het which is optionally substituted on a ring carbon by one or more Q.
In a further aspect of the invention, more preferably R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-4alkyl optionally substituted by one or more Q and a carbon-linked 5-or 6-membered heteroaryl ring containing 1-3 heteroatoms optionally substituted on a ring carbon by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a saturated Het which is optionally substituted on a ring carbon by one or more Q.
In a further aspect of the invention, particularly R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-4alkyl optionally substituted by one or more Q and a carbon-linked 6-membered heteroaryl ring containing 1-2 nitrogen atoms optionally substituted on a ring carbon by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a saturated Het which is optionally substituted on a ring carbon by one or more Q.
Preferably R8 is C1-6alkyl and phenyl optionally substituted by one or more R10.
More preferably R8 is C1-4alkyl and phenyl optionally substituted by one R10.
Particularly R8 is methyl and 4-methoxyphenyl.
Preferably Q is C1-6alkyl-Mxe2x80x94 optionally substituted with one or more R11, R13xe2x80x94Mxe2x80x94, hydroxy, halo, sulphamoyl, amino, carbamoyl or ureido.
More preferably Q is C1-4alkyl-Mxe2x80x94 optionally substituted with one or more R11, R13xe2x80x94Mxe2x80x94, hydroxy, halo, sulphamoyl, amino, carbamoyl or ureido.
Particularly Q is Mexe2x80x94Mxe2x80x94 optionally substituted with one or more R11, R13xe2x80x94Mxe2x80x94, hydroxy, halo, sulphamoyl, amino, carbamoyl or ureido.
More particularly Q is hydroxy or methyl.
Particularly preferred Q is hydroxy.
In another aspect of the invention, preferably Q is hydroxy, acetamido, acetyl, dimethylamino, methoxy or methyl.
Preferably T is xe2x80x94C(O)xe2x80x94.
Preferably M is xe2x80x94Oxe2x80x94, xe2x80x94N(R14)xe2x80x94, xe2x80x94N(R14)C(O)xe2x80x94, xe2x80x94C(O)N(R14)xe2x80x94, xe2x80x94S(O)nxe2x80x94, wherein n is 1-2, xe2x80x94OC(O)xe2x80x94 or xe2x80x94SO2N(R14)xe2x80x94 or M is a direct bond.
In one aspect of the invention, preferably M is xe2x80x94Oxe2x80x94, xe2x80x94N(R14)xe2x80x94, xe2x80x94N(R14)C(O)xe2x80x94, xe2x80x94C(O)N(R14)xe2x80x94, xe2x80x94S(O)nxe2x80x94 wherein n is 1-2, xe2x80x94OC(O)xe2x80x94 or xe2x80x94SO2N(R14)xe2x80x94.
In another aspect of the invention, preferably M is a direct bond.
Preferably D is C1-6alkanoyl, C1-6alkylsulphonyl, Nxe2x80x94(C1-6alkyl)2carbamoyl, phenyl or a carbon linked heterocyclic group.
More preferably D is C1-4alkanoyl, C1-4alkylsulphonyl, Nxe2x80x94(C1-4alkyl)2carbamoyl, phenyl or a carbon linked heterocyclic group.
Particularly D is acetyl, mesyl, N,N-dimethylcarbamoyl, phenyl, pyridyl or pyrimidyl.
More particularly D is acetyl.
Preferably R13 is phenyl or a heterocyclic group.
Preferably R14 is hydrogen or C1-4alkyl optionally substituted by one or more R15 with the proviso that R15 is not a substituent on the carbon attached to the nitrogen atom of M.
Preferably R15 is hydroxy.
Therefore preferably R1 and R2 are each selected independently from hydrogen, phenyl {optionally substituted by one or more methoxy, fluoro, carbamoyl, acetylamino, sulphamoyl, methylsulphinyl, mesyl, methoxycarbonyl, methyl, amino, methylamino, ureido, hydroxy, chloro, acetyl, N-methylcarbamoyl and mesylamino}, C1-6alkyl {optionally substituted by one or more hydroxy, methoxy, N,N-dimethylamino, acetylamino and N,N-dimethylcarbamoyl}, C1-6alkanoyl, and a heterocyclic group {optionally substituted on a ring carbon by one or more hydroxy, methoxy, N,N-dimethylamino and methyl},
or R1 and R2 together with the nitrogen atom to which they are attached form a group Het which is optionally substituted on a ring carbon by one or more hydroxy and wherein if group Het contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from acetyl, mesyl, phenyl, a carbon linked heterocyclic group and N,N-dimethylcarbamoyl.
Therefore more preferably R1 and R2 are each selected independently from hydrogen, C1-6alkyl {optionally substituted by one or more hydroxy}, phenyl {optionally substituted by methoxy} and pyridyl optionally substituted on a ring carbon by methyl,
or R1 and R2 together with the nitrogen atom to which they are attached form a piperidinyl, pyrrolidinyl or azetidinyl ring which is optionally substituted on a ring carbon by one or more hydroxy.
Therefore in one aspect of the invention particularly R1 and R2 are each selected independently from hydrogen, 2-hydroxypropyl, 2-hydroxy-2-methylpropyl, 3-hydroxybutyl,
or R1 and R2 together with the nitrogen atom to which they are attached form a 4-hydroxypiperidinyl, 3-hydroxypiperidinyl, 3-hydroxypyrolidinyl or 3-hydroxyazetidinyl ring.
Therefore in one aspect of the invention more particularly one of R1 and R2 is hydrogen, and the other is selected from 2-hydroxypropyl, 2-hydroxy-2-methylpropyl, 3-hydroxybutyl,
or R1 and R2 together with the nitrogen atom to which they are attached form a 4-hydroxypiperidinyl, 3-hydroxypiperidinyl, 3-hydroxypyrolidinyl or 3-hydroxyazetidinyl ring.
Therefore in another aspect of the invention particularly R1 and R2 are each selected independently from hydrogen, methyl, 2-hydroxypropyl, 4-methoxyphenyl, 5-methylpyridyl,
or R1 and R2 together with the nitrogen atom to which they are attached form a 4-hydroxypiperidinyl ring.
In another aspect of the invention, more particularly R1 and R2 are each selected independently from hydrogen, phenyl, 4-methoxyphenyl, 4-acetylphenyl, 4-acetamidophenyl, 4-dimethylaminophenyl, methyl, ethyl, isopropyl, 4-hydroxycyclohexyl, 1-methyl-2-hydroxyethyl, 1,1-dimethyl-2-hydroxyethyl, 2-hydroxyethyl, 2-hydroxypropyl, 2,3-dihydroxypropyl, pyrid-3-yl and 5-methylpyrid-2-yl,
or R1 and R2 together with the nitrogen atom to which they are attached form 3-hydroxypyrrolidin-1-yl, 3-hydroxypiperidin-1-yl, 4-hydroxypiperidin-1-yl or morpholino.
In a further aspect of the invention, more particularly R1 and R2 are each selected independently from hydrogen, phenyl, 4-methylphenyl, 2-methoxyphenyl, 4-methoxyphenyl, 4-acetylphenyl, 4-acetamidophenyl, 4-dimethylaminophenyl, 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl, methyl, ethyl, isopropyl, 4-hydroxycyclohexyl, 1-methyl-2-hydroxyethyl, 1,1-dimethyl-2-hydroxyethyl, 2-hydroxyethyl, 2-hydroxypropyl, 2,3-dihydroxypropyl, pyrid-3-yl and 5-methylpyrid-2-yl, 2-methoxypyrid-5-yl,
or R1 and R2 together with the nitrogen atom to which they are attached form 3-hydroxypyrrolidin-1-yl, 3-hydroxypiperidin-1-yl, 4-hydroxypiperidin-1-yl or morpholino.
In a further aspect of the invention, more particularly R1 and R2 are each selected independently from hydrogen, methyl, 1-methyl-2-hydroxyethyl or 2-hydroxypropyl.
In a further aspect of the invention, particularly preferred R1 and R2 are each selected independently from hydrogen, methyl, (R,S)-1-methyl-2-hydroxyethyl, (S)-1-methyl-2-hydroxyethyl, (R)-1-methyl-2-hydroxyethyl, (R,S)-2-hydroxypropyl, (S)-2-hydroxypropyl or (R)-2-hydroxypropyl.
In a further aspect of the invention, more particularly preferred one of R1 and R2 is hydrogen and the other is selected from methyl, (R,S)-1-methyl-2-hydroxyethyl, (S)-1-methyl-2-hydroxyethyl, (R)-1-methyl-2-hydroxyethyl, (R,S)-2-hydroxypropyl, (S)-2-hydroxypropyl or (R)-2-hydroxypropyl.
Preferably the R1R2NSO2xe2x80x94 group is ortho to R7.
In one aspect of the invention R3 and R4 are independently Ckalkyl optionally substituted by from 1 to 2k+1 atoms selected from fluoro and chloro wherein k is 1-3.
Preferably R3 and R4 are independently Ckalkyl optionally substituted by from 1 to 2k+1 atoms selected from fluoro and chloro, wherein k is 1-3,
or R3 and R4, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
More preferably R3 and R4 are independently Ckalkyl optionally substituted by from 1 to 2k+1 fluorine atoms, wherein k is 1-2,
or R3 and R4, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
Particularly R3 and R4 are independently methyl, fluoromethyl, difluoromethyl, trifluoromethyl, 2,2,2-trifluoroethyl and perfluoroethyl,
or R3 and R4, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
More particularly R3 and R4 are independently methyl, fluoromethyl, difluoromethyl and trifluoromethyl,
or R3 and R4, together with the carbon atom to which they are attached, form a cyclopropane ring optionally substituted by from 1 to 4 fluorine atoms.
Preferred combinations of R3 and R4 are as follows.
Preferably R3 and R4 are both methyl or one of R3 and R4 is methyl and the other is trifluoromethyl.
More preferably one of R3 and R4 is methyl and the other is trifluoromethyl.
Preferably R5 is selected from halo, haloC1-4alkyl, haloC1-4alkoxy, nitro, C1-4alkyl, C1-4alkoxy, C2-4alkenyl, C2-4alkynyl, hydroxy, hydrogen, amino, cyano, carboxy and sulphamoyl.
More preferably R5 is selected from fluoro, chloro, bromo, nitro, methyl, ethyl, methoxy, ethoxy, ethenyl, ethynyl, hydroxy, hydrogen, amino, cyano, carboxy, sulphamoyl, trifluoromethyl and trifluoromethoxy.
Particularly R5 is selected from fluoro, bromo, chloro, nitro, methyl, ethenyl and ethynyl.
More particularly R5 is selected from fluoro, chloro, bromo and methyl.
Particularly preferred R5 is selected from chloro and bromo.
More particularly preferred R5 is selected from chloro.
In another aspect of the invention, preferably R5 is selected from chloro or methoxy.
Preferably R6 is Q.
In one aspect of the invention preferably R6 is a heterocyclic group {optionally substituted on a ring carbon by one or more hydroxy, acetyl, halo, C1-4alkylsulphonyl, C1-4alkyl, Nxe2x80x94(C1-4alkyl)carbamoyl, Nxe2x80x94(C1-4alkyl)2carbamoyl and wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from C1-4alkyl, C1-4alkylsulphonyl or C1-4alkanoyl}, halo, hydroxy, trifluoromethyl, C1-4alkyl, C1-4alkoxy, C1-4alkylsulphanyl, C1-4alkylsulphinyl, C1-4alkylsulphonyl, C1-4alkylamino, Nxe2x80x94(C1-4alkyl)2amino, C1-4alkanoylamino, (C1-4alkyl)sulphonylamino, Nxe2x80x94(C1-4alkyl)aminosulphonyl, Nxe2x80x94(C1-4alkyl)2aminosulphonyl, Nxe2x80x94(C1-4alkyl)carbamoyl, Nxe2x80x94(C1-4alkyl)2carbamoyl {wherein any C1-4alkyl group may be optionally substituted by one or more hydroxy, methoxy, N,N-dimethylamino, amino, a heterocyclic group [wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from methyl or acetyl], acetylamino and N,N-dimethylcarbamoyl}, (heterocyclic group)sulphanyl, (heterocyclic group)sulphinyl, (heterocyclic group)sulphonyl, N-(heterocyclic group)amino, (heterocyclic group)carbonylamino, (heterocyclic group)sulphonylamino, N-(heterocyclic group)aminosulphonyl, N-(heterocyclic group)Nxe2x80x94(C1-4alkyl)aminosulphonyl, N-(heterocyclic group)carbamoyl, N-(heterocyclic group)Nxe2x80x94(C1-4alkyl)carbamoyl {wherein any heterocyclic group may be optionally substituted on a ring carbon by one or more hydroxy, methoxy, N,N-dimethylamino, amino, a heterocyclic group [wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from methyl, mesyl or acetyl], acetylamino, N,N-dimethylcarbamoyl, 2-hydroxyethylamino, acetyl, methylsulphinyl or methylsulphonyl and wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from methyl, mesyl or acetyl}, phenylsulphanyl, phenylsulphinyl, phenylsulphonyl, N-phenylamino, phenylC1-4alkanoylamino, phenylsulphonylamino, N-phenylaminosulphonyl, N-phenyl-Nxe2x80x94(C1-4alkyl)aminosulphonyl, N-phenylcarbamoyl, N-phenyl-Nxe2x80x94(C1-4alkyl)carbamoyl {wherein any phenyl group may be optionally substituted by one or more hydroxy, methoxy, N,N-dimethylamino, amino, a heterocyclic group [wherein if said heterocyclic group contains an xe2x80x94NHxe2x80x94 moiety that nitrogen may be optionally substituted by a group selected from methyl or acetyl], acetylamino, N,N-dimethylcarbamoyl, 2-hydroxyethylamino, acetyl, methylsulphinyl or methylsulphonyl}.
In one aspect of the invention more preferably R6 is pyrrolidine, piperidine, piperazine (optionally substituted on xe2x80x94NHxe2x80x94 by a group selected from methyl, mesyl and acetyl) or pyridine {which pyrrolidine, piperidine, piperazine or pyridine may be optionally substituted on a ring carbon by one or more hydroxy, acetyl, mesyl, ethylsulphonyl, methyl, ethyl, methoxy, ethoxy, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl}, fluoro, chloro, bromo, hydroxy, trifluoromethyl, methyl, ethyl, methylthlio, ethylthio, methylsulphinyl, ethylsulphinyl, mesyl, ethylsulphonyl, methylamino, ethylamino, N,N-dimethylamino, N,N-diethylamino, N-methyl-N-ethylamino, acetylamino, propionylamino, mesylamino, ethylsulphonylamino, methylaminosulphonyl, ethylaminosulphonyl, N,N-dimethylaminosulphonyl, N,N-diethylaminosulphonyl, N-methyl-N-ethylaminosulphonyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, {wherein any methyl or ethyl may be optionally substituted by one or more hydroxy, methoxy, N,N-dimethylamino, amino, piperazine, N-methylpiperazine, N-acetylpiperazine, morpholine, acetylamino and N,N-dimethylcarbamoyl}, pyridylsulphinyl, pyridylsulphonyl, N-pyridylamino, pyridylcarbonylamino, pyridylsulphonylamino, N-pyridylaminosulphonyl, N-pyridylcarbamoyl {wherein any pyridyl group may be optionally substituted on a ring carbon by one or more hydroxy, methoxy, N,N-dimethylamino, amino, piperazine, N-methylpiperazine, N-acetylpiperazine, morpholine, acetylamino, N,N-dimethylcarbamoyl, 2-hydroxyethylamino, acetyl, methylsulphinyl or methylsulphonyl}, phenylsulphinyl, phenylsulphonyl, N-phenylamino, benzoylamino, phenylsulphonylamino, N-phenylaminosulphonyl, N-phenylcarbamoyl {wherein any phenyl group may be optionally substituted by one or more hydroxy, methoxy, N,N-dimethylamino, amino, piperazine, N-methylpiperazine, N-acetylpiperazine, morpholine, acetylamino, N,N-dimethylcarbamoyl, 2-hydroxyethylamino, acetyl, methylsulphinyl or methylsulphonyl}.
In one aspect of the invention particularly R6 is fluoro, chloro, acetamido and methylsulphanyl.
In another aspect of the invention preferably R6 is hydrogen, halo, hydroxy, carbamoyl, (C1-4alkyl)2amino, C1-4alkanoylamino, (C1-4alkyl)sulphonylamino, Nxe2x80x94(C1-4alkyl)aminosulphonyl, Nxe2x80x94(C1-4alkyl)2aminosulphonyl, Nxe2x80x94(C1-4alkyl)carbamoyl, Nxe2x80x94(C1-4alkyl)2carbamoyl, C1-4alkyloxy, C1-6alkyl, C1-4alkylamino, C1-4alkylsulphinyl, C1-4alkylsulphonyl {wherein any C1-4alkyl may be optionally substituted by one or more halo, hydroxy, amino, carbamoyl, N,N-dimethylcarbamoyl, methylsulphinyl, methylsulphonyl, N-methylpiperazinyl or N-acetylpiperazinyl}, morpholino, piperazinyl {optionally substituted on xe2x80x94NHxe2x80x94 with methyl, 2-hydroxyethyl or acetyl}, (heterocyclic group)sulphinyl, (heterocyclic group)sulphonyl {wherein any heterocyclic group is optionally substituted by one or more halo, hydroxy, 2-hydroxyethylamino, amino, carbamoyl, N,N-dimethylcarbamoyl, methylsulphinyl, methylsulphonyl, N-methylpiperazinyl or N-acetylpiperazinyl}, phenylsulphinyl, phenylsulphonyl {wherein any phenyl is optionally substituted by one or more halo, hydroxy, 2-hydroxyethylamino, amino, carbamoyl, N,N-dimethylcarbamoyl, methylsulphinyl, methylsulphonyl, N-methylpiperazinyl or N-acetylpiperazinyl }.
In another aspect of the invention more preferably R6 is hydrogen, halo, hydroxy, carbamoyl, N,N-dimethylamino, N,N-diethylamino, N-methyl-N-ethylamino, acetylamino, propionylamino, mesylamino, ethylsulphonylamino, methylaminosulphonyl, ethylaminosulphonyl, N,N-dimethylaminosulphonyl, N,N-diethylaminosulphonyl, N-methyl-N-ethylaminosulphonyl, N-methylcarbamoyl, N-ethylcarbamoyl, N,N-dimethylcarbamoyl, N,N-diethylcarbamoyl, N-methyl-N-ethylcarbamoyl, methyl, ethyl, methoxy, ethoxy, methylamino, etlylamino, methylsulphinyl, ethylsulphinyl, mesyl, ethylsulphonyl {wherein any methyl or ethyl may be optionally substituted by one or more halo, hydroxy, amino, carbamoyl, N,N-dimethylcarbamoyl, methylsulphinyl, methylsuphonyl, N-methylpiperazinyl or N-acetylpiperazinyl}, morpholino, piperazinyl {optionally substituted on xe2x80x94NHxe2x80x94 with optionally substituted on xe2x80x94NHxe2x80x94 with methyl, 2-hydroxyethyl or acetyl}, (heterocyclic group)sulphinyl, (heterocyclic group)sulphonyl {wherein any heterocyclic group is optionally substituted by one or more halo, hydroxy, 2-hydroxyethylamino, amino, carbamoyl, N,N-dimethylcarbamoyl, methylsulphinyl, methylsulphonyl, N-methylpiperazinyl or N-acetylpiperazinyl}, phenylsulphinyl, phenylsulphonyl {wherein any phenyl is optionally substituted by one or more halo, hydroxy, 2-hydroxyethylamino, amino, carbamoyl, N,N-dimethylcarbamoyl, methylsulphinyl, methylsulphonyl, N-methylpiperazinyl or N-acetylpiperazinyl }.
In a further aspect of the invention, preferably R6 is chloro.
In a further aspect of the invention, preferably R6 is selected from hydrogen, chloro, iodo, methylsulphanyl or methyl.
In a further aspect of the invention, preferably R6 is selected from chloro, iodo, methylsulphanyl or methyl.
In a further aspect of the invention, preferably R6 is selected from chloro or methyl.
In one aspect of the invention, preferably R6 is ortho to R5.
In another aspect of the invention, preferably R6 is ortho to R7.
Preferably R7 is hydrogen, C1-4alkyl, halo and C1-4alkoxy.
More preferably R7 is hydrogen, methyl, fluoro, chloro and methoxy.
Particularly R7 is hydrogen and fluoro.
More particularly R7 is hydrogen.
In another aspect of the invention, preferably R7 is hydrogen or methoxy.
Where xe2x80x94C(OH)(R3)(R4) represents a chiral center, the (R)-configuration is generally the preferred stereochemistry.
Therefore in a further aspect of the invention there is provided a compound of formula (I) (as depicted above) wherein:
R1 and R2 are each selected independently from hydrogen, phenyl optionally substituted by one or more Q, C3-6cycloalkyl optionally substituted by one or more Q, C1-6alkyl optionally substituted by one or more Q and a heterocyclic group optionally substituted on a ring carbon by one or more Q,
or R1 and R2 together with the nitrogen atom to which they are attached form a group Het which is optionally substituted on a ring carbon by one or more Q;
Q is hydroxy, acetamido, acetyl, dimethylamino, methoxy or methyl;
R3 and R4 is methyl and the other is trifluoromethyl;
R5 is selected from chloro or methoxy;
R6 is selected from hydrogen, chloro, iodo, methylsulphanyl or methyl; and
R7 is hydrogen or methoxy;
or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
Therefore in a further aspect of the invention there is provided a compound of formula (Ixe2x80x2): 
wherein:
Ra is selected from methyl, (R,S)-1-methyl-2-hydroxyethyl, S)-1-methyl-2-hydroxyethyl, (R)-1-methyl-2-hydroxyethyl, (R,S)-2-hydroxypropyl, S)-2-hydroxypropyl or (R)-2-hydroxypropyl; and
R6 is methyl or chloro;
or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
A preferred compound of the invention is any one of the Examples or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
More preferred compounds of the invention are any one of Examples 2, 8, 21, 26, 27, 40, 41 and 43 or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof.
Preferred aspects of the invention are those which relate to the compound or a pharmaceutically acceptable salt thereof.
Within the present invention it is to be understood that a compound of the formula (I) or a salt thereof may exhibit the phenomenon of tautomerism and that the formulae drawings within this specification can represent only one of the possible tautomeric forms. It is to be understood that the invention encompasses any tautomeric form which elevates PDH activity and is not to be limited merely to any one tautomeric form utilized within the formulae drawings. The formulae drawings within this specification can represent only one of the possible tautomeric forms and it is to be understood that the specification encompasses all possible tautomeric forms of the compounds drawn not just those forms which it has been possible to show graphically herein.
It will be appreciated by those skilled in the art that certain compounds of formula (I) contain one or more asymmetrically substituted carbon and/or sulphur atoms, and accordingly may exist in, and be isolated as enantiomerically pure, a mixture of diastereoisomers or as a racemate. Some compounds may exhibit polymorphism. It is to be understood that the present invention encompasses any racemic, optically-active, enantiomerically pure, mixture of diastereoisomers, polymorphic or stereoisomeric form, or mixtures thereof, which form possesses properties useful in the elevation of PDH activity, it being well known in the art how to prepare optically-active forms (for example, by resolution of the racemic form by recrystallization techniques, by synthesis from optically-active starting materials, by chiral synthesis, by enzymatic resolution, (for example WO 9738124), by biotransformation, or by chromatographic separation using a chiral stationary phase) and how to determine efficacy for the elevation of PDH activity by the standard tests described hereinafter.
It is also to be understood that certain compounds of the formula (I) and salts thereof can exist in solvated as well as unsolvated forms such as, for example, hydrated forms. It is to be understood that the invention encompasses all such solvated forms which elevate PDH activity.
A compound of the formula (I), or salt thereof, and other compounds of the invention (as hereinafter defined) may be prepared by any process known to be applicable to the preparation of chemically-related compounds. Such processes include, for example, those illustrated in European Patent Applications, Publication Nos. 0524781, 0617010, 0625516, and in GB 2278054, WO 9323358 and WO 9738124.
Another aspect of the present invention provides a process for preparing a compound of formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, which process (in which variable groups are as defined for formula (I) unless otherwise stated) comprises of:
(a) deprotecting a protected compound of formula (II): 
xe2x80x83where Pg is an alcohol protecting group;
(b) coupling an aniline of formula (III): 
xe2x80x83with an acid of formula (IV): 
xe2x80x83wherein G is a hydroxyl group;
(c) coupling an aniline of formula (III) with an activated acid derivative of formula (IV) wherein G is a hydroxyl group which may be protected as an ester, ether or silyl ether;
(d) reacting a compound of formula (V): 
xe2x80x83where K is a leaving atom or group and G is a hydroxyl group which may be protected as an ester; with an amine of formula (VI):
R1R2NHxe2x80x83xe2x80x83(VI);
and thereafter if necessary:
i) converting a compound of the formula (I) into another compound of the formula (I);
ii) removing any protecting groups; or
iii) forming a pharmaceutically acceptable salt or in vivo hydrolysable ester.
Suitable values for Pg are a benzyl group, a silyl group (for example a trialkylsilyl group or an alkyldiphenylsilyl group) or an acetyl protecting group.
K is a leaving atom or group, suitable values for K are for example a halogen atom such as fluoro or chloro.
Specific conditions of the above reactions are as follows:
Process a)
Suitable reagents for deprotecting an alcohol of formula (II) are for example:
1) when Pg is benzyl:
(i) hydrogen in the presence of palladium/carbon catalyst, i.e. hydrogenolysis; or
(ii) hydrogen bromide or hydrogen iodide;
2) when Pg is a silyl protecting group:
(i) tetrabutylammonium fluoride; or
(ii) aqueous hydrofluoric acid;
3) when Pg is acetyl:
i) mild aqueous base for example lithium hydroxide; or
ii) ammonia or an amine such as dimethylamine.
The reaction can be conducted in a suitable solvent such as ethanol, methanol, acetonitrile, or dimethyl sulphoxide and may conveniently be performed at a temperature in the range of xe2x88x9240 to 100xc2x0 C.
Compounds of formula (II) may be prepared according to the following scheme: 
E is a carboxy protecting group. Suitable values for E include C1-6alkyl, such as methyl and ethyl.
Compounds of formula (IIa) are commercially available compounds, or they are known in the literature, or they are prepared by standard processes known in the art. The synthesis of compounds of formula (III) is described below.
Process b)
An aniline of formula (III) and an acid of formula (IV) may be coupled together in the presence of a suitable coupling reagent. Standard peptide coupling reagents known in the art can be employed as suitable coupling reagents, for example conditions such as those described above for the coupling of (IId) and (III), or for example carbonyldiimidazole and dicyclohexyl-carbodiimide, optionally in the presence of a catalyst such as dimethylaminopyridine or 4-pyrrolidinopyridine, optionally in the presence of a base for example triethylamine, pyridine, or 2,6-di-alkyl-pyridines such as 2,6-lutidine or 2,6-di-tert-butylpyridine. Suitable solvents include dimethylacetamide, dichloromethane, benzene, tetrahydrofuran and dimethylformamide. The coupling reaction may conveniently be performed at a temperature in the range of xe2x88x9240 to 40xc2x0 C.
A compound of formula (III) may be prepared according to Schemes 2) or 3): 
or: 
Compounds of formula (IIIa), (IIIe), (IIIf), (IV) and (VI) are commercially available compounds, or they are known in the literature, or they are prepared by standard processes known in the art.
If the resolved acid of formula (IV) is required it may be prepared by any of the known methods for preparation of optically-active forms (for example, by recrystallization of the chiral salt {for example WO 9738124}, by enzymatic resolution or by chromatographic separation using a chiral stationary phase). For example if an (R)-(+) resolved acid is required it may be prepared by the method of Scheme 2 in World Patent Application Publication No. WO 9738124 for preparation of the (S)-(xe2x88x92) acid, i.e. using the classical resolution method described in European Patent Application Publication No. EP 0524781, also for preparation of the (S)-(xe2x88x92) acid, except that (1S,2R)-norephedrine may be used in place of (S)-(xe2x88x92)-1-phenylethylamine. The chiral acid may also be prepared by using the enzymatic resolution method as described in Tetrahedron Asymmetry, 1999, 10, 679.
Process c)
An aniline of formula (III) may be coupled with an activated acid derivative of formula (IV) for example acid chlorides, acid anhydrides, or phenyl esters, wherein G is a hydroxyl group which may be suitably protected as a stable ester or ether (see above synthesis of formula (IId). This coupling may be achieved optionally in the presence of a base for example triethylamine, pyridine, or 2,6-di-alkylpyridines such as 2,6-lutidine or 2,6-di-tert-butylpyridine or 2,6-di-phenylpyridines. Suitable solvents include dimethylacetamide, dichloromethane, benzene, tetrahydrofuran, and dimethylformamide. The coupling reaction may conveniently be performed at a temperature in the range of xe2x88x9240 to 40xc2x0 C.;
Process d)
A compound of formula (V) may be reacted with an amine of formula (VI) in the presence of a base, for example a tertiary amine such as triethylamine and in the presence of a catalyst for example dimethylaminopyridine. Suitable solvents for the reaction include acetonitrile and dimethylformamide. The reaction is conveniently performed at a temperature in the range of from 0 to 120xc2x0 C.
A compound of formula (V) may be prepared according to the following scheme: 
If not commercially available, the necessary starting materials for the procedures such as that described above may be made by procedures which are selected from standard organic chemical techniques, techniques which are analogous to the synthesis of known, structurally similar compounds, or techniques which are analogous to the above described procedure or the procedures described in the examples.
For example, it will be appreciated that certain of the optional aromatic substituents in the compounds of the present invention may be introduced by standard aromatic substitution reactions or generated by conventional functional group modifications or interconversions either prior to or immediately following the processes mentioned above, and as such are included in the process aspect of the invention. Such reactions and modifications include, for example, introduction of a substituent by means of an aromatic substitution reaction, reduction of substituents alkylation of substituents and oxidation of substituents. The reagents and reaction conditions for such procedures are well known in the chemical art. Particular examples of aromatic substitution reactions include the introduction of a nitro group using concentrated nitric acid, the introduction of an acyl group using, for example, an acylhalide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; the introduction of an alkyl group using an alkyl halide and Lewis acid (such as aluminium trichloride) under Friedel Crafts conditions; and the introduction of a halogeno group. Particular examples of modifications include the reduction of a nitro group to an amino group by, for example, catalytic hydrogenation with a nickel catalyst or treatment with iron in the presence of hydrochloric acid with heating; oxidation of alkylthio to alkylsulphinyl or alkylsulphonyl using, for example, hydrogen peroxide in acetic acid with heating or 3-chloroperbenzoic acid. Particular examples of functional group interconversions are for example conversion of an aniline into a halophenyl by, for example, diazotisation in the presence of cupurous halides.
It is noted that many of the starting materials for synthetic methods as described above are commercially available and/or widely reported in the scientific literature, or could be made from commercially available compounds using adaptations of processes reported in the scientific literature.
It will also be appreciated that in some of the reactions mentioned herein it may be necessary/desirable to protect any sensitive groups in the compounds. The instances where protection is necessary or desirable and suitable methods for protection are known to those skilled in the art. Thus, if reactants include groups such as amino, carboxy or hydroxy it may be desirable to protect the group in some of the reactions mentioned herein.
A suitable protecting group for an amino or alkylamino group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an alkoxycarbonyl group, for example a methoxycarbonyl, ethoxycarbonyl or t-butoxycarbonyl group, an arylmethoxycarbonyl group, for example benzyloxycarbonyl, or an aroyl group, for example benzoyl. The deprotection conditions for the above protecting groups necessarily vary with the choice of protecting group. Thus, for example, an acyl group such as an alkanoyl or alkoxycarbonyl group or an aroyl group may be removed for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an acyl group such as a t-butoxycarbonyl group may be removed, for example, by treatment with a suitable acid such as, for example hydrochloric sulphuric or phosphoric acid or trifluoroacetic acid and an arylmethoxycarbonyl group such as a benzyloxycarbonyl group may be removed, for example, by hydrogenation in the presence of a catalyst such as palladium-on-carbon, or by treatment with a Lewis acid for example boron tris(trifluoroacetate). A suitable alternative protecting group for a primary amino group is, for example, a phthaloyl group which may be removed by treatment with an alkylamine, for example dimethylaminopropylamine, or with hydrazine.
A suitable protecting group for a hydroxy group is, for example, an acyl group, for example an alkanoyl group such as acetyl, an aroyl group, for example benzoyl, or an arylmethyl group, for example benzyl. The deprotection conditions for the above protecting groups will necessarily vary with the choice of protecting group. Thus, for example an acyl group such as an alkanoyl or an aroyl group may be removed, for example, by hydrolysis with a suitable base such as an alkali metal hydroxide, for example lithium or sodium hydroxide. Alternatively an arylmethyl group such as a benzyl group may be removed, for example, by hydrogenation in the presence of a catalyst such as palladium-on-carbon.
A suitable protecting group for a carboxy group is, for example, an esterifying group, for example a methyl or an ethyl group which may be removed, for example, by hydrolysis with a base such as sodium hydroxide, or for example a t-butyl group which may be removed, for example, by treatment with an acid, for example an organic acid such as trifluoroacetic acid, or for example a benzyl group which may be removed, for example, by hydrogenation over a catalyst such as palladium-on-carbon.
The protecting groups may be removed at any convenient stage in the synthesis using conventional techniques well known in the chemical art.
In cases where compounds of formula (I) are sufficiently basic or acidic to form stable acid or basic salts, administration of the compound as a salt may be appropriate, and pharmaceutically acceptable salts may be made by conventional methods such as those described following. Examples of suitable pharmaceutically acceptable salts are organic acid addition salts formed with acids which form a physiologically acceptable anion, for example, tosylate, methanesulphonate, acetate, tartrate, citrate, succinate, benzoate, ascorbate, xcex1-ketoglutarate, and xcex1-glycerophosphate. Suitable inorganic salts may also be formed such as sulphate, nitrate, and hydrochloride.
Pharmaceutically acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound of formula (I) (or its ester) with a suitable acid affording a physiologically acceptable anion. It is also possible with most compounds of the invention to make a corresponding alkali metal (e.g. sodium, potassium, or lithium) or alkaline earth metal (e.g. calcium) salt by treating a compound of formula (I) (and in some cases the ester) with one equivalent of an alkali metal or alkaline earth metal hydroxide or alkoxide (e.g. the ethoxide or methoxide) in aqueous medium followed by conventional purification techniques.
The compounds of the formula (I) may be administered in the form of a prodrug which is broken down in the human or animal body to give a compound of the formula (I). Examples of prodrugs include in vivo hydrolysable esters of a compound of the formula (I).
An in vivo hydrolysable ester of a compound of the formula (I) containing carboxy or hydroxy group is, for example, a pharmaceutically acceptable ester which is hydrolysed in the human or animal body to produce the parent acid or alcohol.
Suitable in vivo hydrolysable esters for a compound of the formula (I) containing a carboxy group include C1-6alkoxymethyl esters for example methoxymethyl, C1-6alkanoyloxymethyl esters for example pivaloyloxymethyl, phthalidyl esters, C3-8cycloalkoxycarbonyloxyC1-6alkyl esters for example 1-cyclohexylcarbonyloxyethyl; 1,3-didxolen-2-onylmethyl esters for example 5-methyl-1,3-dioxolen-2-onylmethyl; and C1-6alkoxycarbonyloxyethyl esters for example 1-methoxycarbonyloxyethyl and may be formed at any carboxy group in the compounds of this invention.
Suitable in vivo hydrolysable esters of a compound of the formula (I) containing a hydroxy group includes inorganic esters such as phosphate esters and xcex1-acyloxyalkyl ethers. Examples of xcex1-acyloxyalkyl ethers include acetoxymethoxy and 2,2-dimethylpropionyloxymethoxy. Other in vivo hydrolysable ester forming groups for hydroxy include alkanoyl, benzoyl, phenylacetyl and substituted benzoyl and phenylacetyl, alkoxycarbonyl (to give alkyl carbonate esters), dialkylcarbamoyl and N-(dialkylaminoethyl)-N-alkylcarbamoyl (to give carbamates), dialkylaminoacetyl and carboxyacetyl. Examples of substituents for benzoyl include morpholino and piperazino linked from a ring nitrogen atom via a methylene group to the 3- or 4- position of the benzoyl ring.
In vivo cleavable prodrugs of compounds of formula (I) also include in vivo hydrolysable amides of compounds of the formula (I) containing a carboxy group, for example, a Nxe2x80x94C1-6alkyl or Nxe2x80x94dixe2x80x94C1-6alkyl amide such as N-methyl, N-ethyl, N-propyl, N-dimethyl, N-ethyl-N-methyl or N-diethyl amide.
The identification of compounds which elevate PDH activity is the subject of the present invention. These properties may be assessed, for example, using one or more of the procedures set out below:
(a) In vitro Elevation of PDH Activity
This assay determines the ability of a test compound to elevate PDH activity. cDNA encoding PDH kinase may be obtained by Polymerase Chain Reaction (PCR) and subsequent cloning. This may be expressed in a suitable expression system to obtain polypeptide with PDH kinase activity. For example rat PDHkinaseII (rPDHKII) obtained by expression of recombinant protein in Escherichia coli (E. Coli), was found to display PDH kinase activity.
In the case of the rPDHKII (Genbank accession number U10357) a 1.3 kb fragment encoding the protein was isolated by PCR from rat liver cDNA and cloned into a vector (for example pQE32xe2x80x94Quiagen Ltd.). The recombinant construct was transformed into E. coli (for example M15pRep4xe2x80x94Quiagen Ltd.). Recombinant clones were identified, plasmid DNA was isolated and subjected to DNA sequence analysis. One clone which had the expected nucleic acid sequence was selected for the expression work. Details of the methods for the assembly of recombinant DNA molecules and the expression of recombinant proteins in bacterial systems can be found in standard texts for example Sambrook et al, 1989, Molecular Cloning xe2x80x94A Laboratory Manual, 2nd edition, Cold Spring Harbour Laboratory Press. Other known PDH kinases for use in assays, may be cloned and expressed in a similar manner.
For expression of rPDHKII activity, E. coli strain M15pRep4 cells were transformed with the pQE32 vector containing rPDHKII cDNA. This vector incorporates a 6-His tag onto the protein at its N-terminus. E. coli were grown to an optical density of 0.6 (600 nM) and protein expression was induced by the addition of 10 xcexcM isopropylthio-xcex2-galactosidase. Cells were grown for 18 hours at 18xc2x0 C. and harvested by centrifugation. The resuspended cell paste was lysed by homogenisation and insoluble material removed by centrifugation at 24000xc3x97g for 1 hour. The 6-His tagged protein was removed from the supernatant using a nickel chelating nitrilotriacetic acid resin (Ni-NTA: Quiagen Ltd.) matrix (Quiagen) which was washed with 20 mM tris(hydroxymethyl)aminomethane-hydrogen chloride, 20 mM imidazole, 0.5 M sodium chloride pH 8.0, prior to elution of bound protein using a buffer containing 20 mM tris(hydroxymethyl)aminomethane-hydrogen chloride, 200 mM imidazole, 0.15 M sodium chloride pH 8.0. Eluted fractions containing 6-His protein were pooled and stored in aliquots at xe2x88x9280xc2x0 C. in 10% glycerol.
Each new batch of stock enzyme was titrated in the assay to determine a concentration giving approximately 90% inhibition of PDH in the conditions of the assay. For a typical batch, stock enzyme was diluted to 7.5 xcexcg/ml.
For assay of the activity of novel compounds, compounds were diluted with 10% dimethyl sulphoxide (DMSO) and 10 xcexcl transferred to individual wells of 96-well assay plates. Control wells contained 20 xcexcl 10% DMSO instead of compound. 40 xcexcl Buffer containing 50 mM potassium phosphate buffer pH 7.0, 10 mM ethylene glycol-bis(xcex2-aminoethyl ether)-N,N-tetracetic acid (EGTA), 1 mM benzamidine, 1 mM phenylmethylsulphonyl fluoride (PMSF), 0.3 mM tosyl-L-lysine chloromethyl ketone (TLCK), 2 mM dithiothreitol (DTT), recombinant rPDHKII and compounds were incubated in the presence of PDH kinase at room temperature for 45 minutes. In order to determine the maximum rate of the PDH reaction a second series of control wells were included containing 10% DMSO instead of compound and omitting rPDHKII. PDH kinase activity was then initiated by the addition of 5 xcexcM ATP, 2 mM magnesium chloride and 0.04 U/ml PDH (porcine heart PDH Sigma P7032) in a total volume of 50 xcexcl and plates incubated at ambient temperature for a further 45 minutes. The residual activity of the PDH was then determined by the addition of substrates (2.5 mM coenzyme A, 2.5 mM thiamine pyrophosphate (cocarboxylase), 2.5 mM sodium pyruvate, 6 mM NAD in a total volume of 80 xcexcl and the plates incubated for 90 minutes at ambient temperature. The production of reduced NAD (NADH) was established by measured optical density at 340 nm using a plate reading spectrophotometer. The ED50 for a test compound was determined in the usual way using results from 12 concentrations of the compound.
(b) In vitro Elevation of PDH Activity in Isolated Primary Cells
This assay determines the ability of compounds to stimulate pyruvate oxidation in primary rat hepatocytes.
Hepatocytes were isolated by the two-step collagenase digestion procedure described by Seglen (Methods Cell Biol. (1976) 13, 29-33) and plated out in 6-well culture plates (Falcon Primaria) at 600000 viable cells per well in Dulbecco""s Modified Eagles Medium (DMEM, Gibco BRL) containing 10% foetal calf serum (FCS), 10% penicillin/streptomycin (Gibco BRL) and 10% non-essential amino acids (NEAA, Gibco BRL). After 4 hours incubation at 37xc2x0 C. in 5% CO2, the medium was replaced with Minimum Essential Medium (MEM, Gibco BRL) containing NEAA and penicillin/streptomycin as above in addition to 10 nM dexamethasone and 10 nM insulin.
The following day cells were washed with phosphate buffered saline (PBS) and medium replaced with 1 ml HEPES-buffered Krebs solution (25 mM HEPES, 0.15 M sodium chloride, 25 mM sodium hydrogen carbonate, 5 mM potassium chloride, 2 mM calcium chloride, 1 mM magnesium sulphate, 1 mM potassium dihydrogen phosphate) containing the compound to be tested at the required concentration in 0.1% DMSO. Control wells contained 0.1% DMS0 only and a maximum response was determined using a 10 xcexcM treatment of a known active compound. After a preincubation period of 40 minutes at 37xc2x0 C. in 5% CO2, cells were pulsed with sodium pyruvate to a final concentration of 0.5 mM (containing 1-14C sodium pyruvate (Amersham product CFA85) 0.18 Ci/mmole) for 12 minutes. The medium was then removed and transferred to a tube which was immediately sealed with a bung containing a suspended centre well. Absorbent within the centre well was saturated with 50% phenylethylamine, and CO2 in the medium released by the addition of 0.2 xcexcl 60% (w/v) perchloric acid (PCA). Released 14CO2 trapped in the absorbent was determined by liquid scintillation counting. The ED50 for a test compound was determined in the usual way using results from 7 concentrations of the compound.
(c) In vivo Elevation of PDH Activity
The capacity of compounds to increase the activity of PDH in relevant tissues of rats may be measured using the test described hereinafter. Typically an increase in the proportion of PDH in its active, nonphosphorylated form may be detected in muscle, heart, liver and adipose tissue after a single administration of an active compound. This may be expected to lead to a decrease in blood glucose after repeated administration of the compound. For example a single administration of DCA, a compound known to activate PDH by inhibition of PDH kinase (Whitehouse, Cooper and Randle (1974) Biochem. J. 141, 761-774) 150 mg/kg, intraperitoneally, increased the proportion of PDH in its active form (Vary et al. (1988) Circ. Shock 24, 3-18) and after repeated administration resulted in a significant decrease in plasma glucose (Evans and Stacpoole (1982) Biochem. Pharmacol.31, 1295-1300).
Groups of rats (weight range 140-180 g) are treated with a single dose or multiple doses of the compound of interest by oral gavage in an appropriate vehicle. A control group of rats is treated with vehicle only. At a fixed time after the final administration of compound, animals are terminally anaesthetised, tissues are removed and frozen in liquid nitrogen. For determination of PDH activity, muscle samples are disrupted under liquid nitrogen prior to homogenisation by one thirty-second burst in a Polytron homogenizer in 4 volumes of a buffer containing 40 mM potassium phosphate pH 7.0, 5 mM EDTA, 2 mM DTT, 1% Triton X-100, 10 mM sodium pyruvate, 10 xcexcM phenylmethylsulphonyl chloride (PMSF) and 2 xcexcg/ml each of leupeptin, pepstain A and aprotinin. Extracts are centrifuged before assay. A portion of the extract is treated with PDH phosphatase prepared from pig hearts by the method of Siess and Wieland (Eur. J. Biochem (1972) 26, 96):20 xcexcl extract, 40 xcexcl phosphatase (1:20 dilution), in a final volume of 125 xcexcl containing 25 mM magnesium chloride, 1 mM calcium chloride. The activity of the untreated sample is compared with the activity of the dephosphorylated extract thus prepared. PDH activity is assayed by the method of Stansbie et al., (Biochem. J. (1976) 154, 225). 50 xcexcl Extract is incubated with 0.75 mM NAD, 0.2 mM CoA, 1.5 mM thiamine pyrophosphate (TPP) and 1.5 mM sodium pyruvate in the presence of 20 xcexcg/ml p-(p-amino-phenylazo) benzene sulphonic acid (AABS) and 50 mU/ml arylamine transferase (AAT) in a buffer containing 100 mM tris(hydroxymethyl)aminomethane, 0.5 mM EDTA, 50 mM sodium fluoride, 5 mM 2-mercaptoethanol and 1 mM magnesium chloride pH 7.8. AAT is prepared from pigeon livers by the method of Tabor et al. (J. Biol. Chem. (1953) 204, 127). The rate of acetyl CoA formation is determined by the rate of reduction of AABS which is indicated by a decrease in optical density at 460 nm.
Liver samples are prepared by an essentially similar method, except that sodium pyruvate is excluded from the extraction buffer and added to the phosphatase incubation to a final concentration of 5 mM.
Treatment of an animal with an active compound results in an increase in the activity of PDH complex in tissues. This is indicated by an increase in the amount of active PDH (determined by the activity of untreated extract as a percentage of the total PDH activity in the same extract after treatment with phiosphatase).
According to a further aspect of the invention there is provided a pharmaceutical composition which comprises a, compound of the formula (I) as defined hereinbefore or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, in association with a pharmaceutically acceptable excipient or carrier.
The composition may be in a form suitable for oral administration, for example as a tablet or capsule, for parenteral injection (including intravenous, subcutaneous, intramuscular, intravascular or infusion) for example as a sterile solution, suspension or emulsion, for topical administration for example as an ointment or cream or for rectal administration for example as a suppository. In general the above compositions may be prepared in a conventional manner using conventional excipients.
The compositions of the present invention are advantageously presented in unit dosage form. The compound will normally be administered to a warm-blooded animal at a unit dose within the range 5-5000 mg per square meter body area of the animal, i.e. approximately 0.1-100 mg/kg. A unit dose in the range, for example, 1-100 mg/kg, preferably 1-50 mg/kg is envisaged and this normally provides a therapeutically-effective dose. A unit dose form such as a tablet or capsule will usually contain, for example 1-250 mg of active ingredient.
According to a further aspect of the present invention there is provided a compound of the formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof as defined hereinbefore for use in a method of treatment of the human or animal body by therapy.
We have found that compounds of the present invention elevate PDH activity and are therefore of interest for their blood glucose-lowering effects.
A further feature of the present invention is a compound of formula (I) and pharmaceutically acceptable salts or in vivo hydrolysable esters thereof for use as a medicament.
Conveniently this is a compound of formula (I), or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, for use as a medicament for producing an elevation of PDH activity in a warm-blooded animal such as a human being.
Particularly this is a compound of formula (I), or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, for use as a medicament for treating diabetes mellitus in a warm-blooded animal such as a human being.
In another aspect of the invention, particularly this is a compound of formula (I), or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof, for use as a medicament for treating diabetes mellitus, peripheral vascular disease and myocardial ischaemia in a warm-blooded animal such as a human being.
Thus according to a further aspect of the invention there is provided the use of a compound of the formula (I), or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof in the manufacture of a medicament for use in the production of an elevation of PDH activity in a warm-blooded animal such as a human being
Thus according to a further aspect of the invention there is provided the use of a compound of the formula (I), or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof in the manufacture of a medicament for use in the treatment of diabetes mellitus in a warm-blooded animal such as a human being.
Thus according to a further aspect of the invention there is provided the use of a compound of the formula (I), or a pharmaceutically acceptable salt or an in Vivo hydrolysable ester thereof in the manufacture of a medicament for use in the treatment of diabetes mellitus, peripheral vascular disease and myocardial ischaemia in a warm-blooded animal such as a human being.
According to a further feature of the invention there is provided a method for producing an elevation of PDH activity in a warm-blooded animal, such as a human being, in need of such treatment which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof as defined hereinbefore.
According to a further feature of the invention there is provided a method of treating diabetes mellitus in a warm-blooded animal, such as a human being, in need of such treatment which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or an in vivo hydrolysable ester thereof as defined hereinbefore.
According to a further feature of the invention there is provided a method of treating diabetes mellitus, peripheral vascular disease and myocardial ischaemia in a warm-blooded animal, such as a human being, in need of such treatment which comprises administering to said animal an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt or an its vivo hydrolysable ester thereof as defined hereinbefore.
As stated above the size of the dose required for the therapeutic or prophylactic treatment of a particular disease state will necessarily be varied depending on the host treated, the route of administration and the severity of the illness being treated. Preferably a daily dose in the range of 1-50 mg/kg is employed.However the daily dose will necessarily be varied depending upon the host treated the particular route of administration, and the severity of the illness being treated. Accordingly the optimum dosage may be determined by the practitioner who is treating any particular patient.
The elevation of PDH activity described herein may be applied as a sole therapy or may involve, in addition to the subject of the present invention, one or more other substances and/or treatments. Such conjoint treatment may be achieved by way of the simultaneous, sequential or separate administration of the individual components of the treatment. For example in the treatment of diabetes mellitus chemotherapy may include the following main categories of treatment:
i) insulin;
ii) insulin secretagogue agents designed to stimulate insulin secretion (for example glibenclamide, tolbutamide, other sulphonylureas);
iii) oral hypoglycaemic agents such as metformin, thiazolidinediones;
iv) agents designed to reduce the absorption of glucose from the intestine (for example acarbose);
v) agents designed to treat complications of prolonged hyperglycaemia;
vi) other agents used to treat lactic acidaemia;
vii) inhibitors of fatty acid oxidation;
viii) lipid lowering agents;
ix) agents used to treat coronary heart disease and peripheral vascular disease such as aspirin, pentoxifylline, cilostazol; and/or
x) thiamine.
As stated above the compounds defined in the present invention are of interest for their ability to elevate the activity of PDH. Such compounds of the invention may therefore be useful in a range of disease states including diabetes mellitus, peripheral vascular disease, (including intermittent claudication), cardiac failure and certain cardiac myopathies, myocardial ischaemia, cerebral ischaemia and reperfusion, muscle weakness, hyperlipidaemias, Alzheimers disease and/or atherosclerosis. Alternatively such compounds of the invention may be useful in a range of disease states including peripheral vascular disease, (including intermittent claudication), cardiac failure and certain cardiac myopathies, myocardial ischaemia, cerebral ischaemia and reperfusion, muscle weakness, hyperlipidaemias, Alzheimers disease and/or atherosclerosis in particular peripheral vascular disease and myocardial ischaemia.
In addition to their use in therapeutic medicine, the compounds of formula (I) and their pharmaceutically acceptable salts are also useful as pharmacological tools in the development and standardisation of in vitro and in vivo test systems for the evaluation of the effects of elevators of PDH activity in laboratory animals such as cats, dogs, rabbits, monkeys, rats and mice, as part of the search for new therapeutic agents.
The invention will now be illustrated by the following non-limiting examples in which, unless stated otherwise:
(i) temperatures are given in degrees Celsius (xc2x0 C.); operations were carried out at room or ambient temperature, that is, at a temperature in the range of 18-25xc2x0 C.;
(ii) organic solutions were dried over anhydrous magnesium sulphate; evaporation of solvent was carried out using a rotary evaporator under reduced pressure (600-4000 Pascals; 4.5-30 mm Hg) with a bath temperature of up to 60xc2x0 C.;
(iii) chromatography unless otherwise stated means flash chromatography on silica gel; thin layer chromatography (TLC) was carried out on silica gel plates; where a xe2x80x9cBond Elutxe2x80x9d column is referred to, this means a column containing 10 g or 20 g of silica of 40 micron particle size, the silica being contained in a 60 ml disposable syringe and supported by a porous disc, obtained from Varian, Harbor City, Calif., USA under the name xe2x80x9cMega Bond Elut SIxe2x80x9d
(iv) in general, the course of reactions was followed by TLC and reaction times are given for illustration only;
(v) yields are given for illustration only and are not necessarily those which can be obtained by diligent process development; preparations were repeated if more material was required; (vi) when given, 1H NMR data is quoted and is in the form of delta values for major diagnostic protons, given in parts per million (ppm) relative to tetramethylsilane (TMS) as an internal standard, determined at 300 MHz using perdeuterio dimethyl sulphoxide (DMSO-d6) as the solvent unless otherwise stated; coupling constants (J) are given in Hz;
(vii) chemical symbols have their usual meanings; SI units and symbols are used;
(viii) solvent ratios are given in percentage by volume;
(ix) mass spectra (MS) were run with an electron energy of 70 electron volts in the chemical ionisation (CI) mode using a direct exposure probe; where indicated ionisation was effected by electron impact (EI) or fast atom bombardment (FAB); where values for m/z are given, generally only ions which indicate the parent mass are reported, and unless otherwise stated the mass ion quoted is the negative mass ion xe2x80x94(Mxe2x80x94H)xe2x88x92;
(x) the following abbreviations are used:
DMSO dimethyl sulphoxide;
DMF N-dimethylformamide;
DCM dichloromethane; and
EtOAc ethyl acetate; and
(xi) where a Chem Elut column is referred to, this means a xe2x80x9cHydromatrixxe2x80x9d extraction cartridge for adsorption of aqueous material, i.e. a polypropylene tube containing a special grade of flux-calcined, high purity, inert diatomaceous earth, pre-buffered to pH 4.5 or 9.0, incorporating a phase-separation filtering material, used according to the manufacturers instructions, obtained from Varian, Harbor City, Calif., USA under the name of xe2x80x9cExtube, Chem Elutxe2x80x9d; xe2x80x9cExtubexe2x80x9d is a registered trademark of International Sorbent Technology Limited;
(xii) where (R) or (S) stereochemistry is quoted at the beginning of a name, unless further clarified, it is to be understood that the indicated stereochemistry refers to the NHxe2x80x94C(O)xe2x80x94C*(OH)(R3)(R4) centre as depicted in formula (I).